The invention relates to materials and methods for the control of seed germination and seedling growth and, more specifically, to the regulation of the gene products of the gibberellin biosynthetic pathway and restoration of normal seed germination and seedling growth by treatment with exogenously applied gibberellins or gibberellin precursors.
Most agriculturally important crop plants are propagated by seed. The seed is planted and under favorable environmental conditions, the seed germinates and grows into crop plants. However, frequently conditions can occur in which after planting of the seed, the seed fails to germinate or germinates poorly producing a thin stand of plants with reduced yield or necessitating the replanting of the crop with new seed at considerable expense to the grower. This has been shown to occur with soybean, corn, and canola in wet and cool field conditions (Wang et al., Enviro. Exp. Bot. 36: 377-383, 1996; Zheng et al., Crop Sci. 34: 1589-1593, 1994). It is often necessary to plant more seeds than predicted to be necessary to achieve a good crop. The percent of seeds that germinate is considered good at 80%. A measurable savings in resources can be achieved if the seed germination can be controlled to achieve 90% or greater seed germination and vigorous seedling growth. Also, seeds may germinate precociously if the environmental conditions at crop maturity are such that the seed prematurely sprout. This is a problem in some wheat varieties and causes a loss of yield and quality of the harvested grain. Dormancy of seeds during storage is an important criteria for a quality product. Adequate storage and shipping characteristics of seeds is an important prerequisite for distributing food products around the world. In many developing countries storage facilities are inadequate and seed and food quality may be affected when seed dormancy is broken and the process of seed germination begins in storage. However, seeds that are chemically treated to inhibit seed germination often show characteristic traits such as reduced plant height and seedling vigor for some period of time after germination. Seed, genetically engineered for a high level of seed dormancy, can be stored more efficiently and suffer fewer side effects than chemically treated germination inhibition.
The failure of seeds to germinate uniformly and at high frequency is an important factor affecting crop yield. Soybean (Glycine max) is a crop species that suffers from loss of seed germination during storage and fails to germinate when soil temperatures are cool. It has been shown that the exogenous treatment of gibberellic acid will stimulate soybean seed germination under conditions that the seed will not normally germinate (Zhang et al., Plant Soil 188: 329-335, 1997). Sugar beet (Beta vulgaris) seed is often chemically treated to improve germination and plant stand which has a direct affect on the yield of the crop. Canola seed germination and seedling growth can be improved at low temperatures by treatment with gibberellic acid (Zheng et al., Crop Sci. 34: 1589-1593, 1994). Improved seedling vigor is observed by these treatments with the plants emerging more quickly from the soil and are more likely to establish themselves under adverse environmental conditions.
There is a need for an effective system which would couple genetically improved seed dormancy with a chemical seed treatment to induce seed germination when germination is desired. The genetic control of gibberellin activity in developing seeds, germinating seeds and during early seedling growth coupled with exogenous replacement of the activity would be an effective means to control seed germination and seedling growth.
Inhibitors of gibberellin biosynthesis suggest that de novo synthesis of GA is a prerequisite for the release from dormancy (Thomas, Plant Growth Reg. 11: 239-248, 1992). A key enzyme of gibberellin biosynthesis is copalyl diphosphate synthase (CPS) (formerly ent-kaurene synthetase (EKS-A)). Two enzymes, CPS and KS-B (ent-kaurene synthetase-B), catalyze the cyclization of geranylgeranyl diphosphate to ent-kaurene. CPS is the first committed step in GA biosynthesis. Plant mutants blocked at CPS show strong adverse germination/seedling vigor phenotypes that can be reversed by the application of an exogenous supply of GA. Although these mutants demonstrate the role of GA in seed germination, they do not establish the developmental timing required for expression of GA for normal seed germination and seedling growth. It has not been previously established that soybean plants require de novo biosynthesis of GA for normal seed germination and early seedling growth. It has also not been previously demonstrated that endogenous levels of GA can be affected by the expression of an antisense RNA to a gene important in the biosynthesis of GA in soybean. There are no known soybean mutants blocked in GA biosynthesis; therefore, the requirement for de novo GA biosynthesis in soybean is unknown. Inhibitors of GA biosynthesis offer a method to investigate the effect of decreased GA biosynthesis on soybean germination and seedling growth. GA biosynthesis inhibitors can block ent-kaurene biosynthesis or can block at ent-kaurene oxidation or can inhibit the late dioxygenase-catalyzed steps (Jung et al., J. Plant Growth Regul. 4: 181-188, 1986).
Dioxygenase enzymes modify various gibberellin substrates. The dioxygenases, 20-oxidase and 3xcex2-hydroxylase, are involved in the biosynthesis of GA precursors and active forms. The overexpression or suppression of the GA 20-oxidase genes affect seedling growth differentially (Hedden, et al., In Genetic and environmental manipulation of horticultural crops. Cockshull, Gray, Seymour and Thomas eds. CAB International 1998). Degradation of bioactive GA in specific tissues of the developing seed, germinating seed and early seeding growth can also regulate GA tissue responses. Genes from Arabidopsis and Phaseolus coccineus have been identified that encode for enzymes that have gibberellin 2-oxidase activity (Thomas, et al., Proc. Natl. Acad. Sci. U.S.A. 96: 4698-4703, 1999).
Pathways which use substrates in common with the gibberellin pathway are known. The carotenoid pathway (Encyclopedia of Plant Physiology. Secondary Plant Products Vol 8: 259, Bell and Charlwood eds.), the phytol pathway (Encyclopedia of Plant Physiology. Secondary Plant Products Vol 8: 207, Bell and Charlwood eds. ) and the gibberellin pathway each use geranylgeranyl pyrophosphate as a key precursor to the synthesis of their respective end products.
The methods of plant biotechnology provide means to express gene products in plants at particular developmental plant growth stages. Gene promoters that express during seed germination and early seedling development are a preferred embodiment of this invention. The present invention provides a method to genetically suppress seed germination and early seedling development, then by necessity restore normal germination with exogenous application of GA compounds to the seed or seedling. The present invention provides genetically-engineered gibberellin-deficient plants. In agriculture, there exists a need for improved materials and methods for the control of seed germination and seedling growth.
The present invention provides materials and methods for the control of seed germination and seedling growth through the use of plants that have altered levels of a hormone such as a gibberellin (GA) that affects seed germination and seedling growth. Such plants can be germinated and grown to maturity by treating the plants, or seeds or seedlings of such plants, a compound that restores substantially normal levels of the hormone or that has hormone activity.
According to one aspect of the invention, methods are provided for growing a transgenic plant that has a transgene that includes a promoter and, operably linked to the promoter, a sequence that, when expressed, alters the level of a hormone, for example GA. The transgene thus causes one or more abnormal phenotypes in the transgenic plant or seeds or seedlings thereof, such as a shortened hypocotyl, shortened epicotyl, or both (compared with a control, i.e., an otherwise identical except for lacking the transgene). A phenotypically normal plant can be grown from the transgenic plant after applying to the plant or to the seed or seedling thereof (for example, applying indirectly to soil or directly to the plant, seed or seedling) a composition that includes a first compound that is metabolized to produce a second compound that substantially eliminates the abnormal phenotype. In the case of GA-deficient plants, for example, use of GA precursors or biosynthetic intermediates (e.g., ent-kaurene, ent-kaurenoic acid, ent-7xcex1-hydroxykaurenoic acid, steviol, GA12-aldehyde, GA12, GA15, GA24, GA9, GA53, GA44, GA19, GA20, GA5, and GA3-3-acetate) helps to properly regulate the amount of bioactive GA that is available within the plants, seeds, or seedlings. Preferred compounds for administration to GA-deficient plants include GA9, GA15, GA19, GA24, GA44, GA53, GA5, and steviol. Several approaches are described herein for producing GA-deficient plants for which the preferred promoter is preferentially expressed in developing seeds, during seed germination, or in young seedlings.
According to one approach, such methods involve the use of transgenic plants having altered hormone levels resulting from a transgene that comprises a sequence that, when expressed, reduces expression of an enzyme in the pathway for biosynthesis of the hormone. For example, the sequence may be in an antisense orientation (i.e., an antisense construct), or suppress hormone biosynthesis as a ribozyme, triplex DNA, by cosuppression, or by any other well-known methods for reducing the expression of endogenous plant genes. For example, in order to alter GA levels, the sequence may suppress expression of an enzyme such as a copalyl diphosphate synthase, a 3xcex2-hydroxylase, or a C-20 oxidase, such as by antisense expression of a sequence that comprises at least 12 contiguous nucleotides (and preferably at least 15, 18, 20, 24, 30, 40, or longer, up to and including the entire length of) SEQ ID NO:1, 2, 3, 4, 5, 6, or 8 or complements thereof, or, alternatively, a sequence that hybridizes under high stringency conditions to SEQ ID NO:1, 2, 3, 4, 5, 6, or 8 or complements thereof.
According to another approach, such methods involve the use of transgenic plants having altered hormone levels resulting from a transgene that comprises a sequence that inactivates the hormone. For example, plants having altered levels of a GA can be produced by expression in the plants of a sequence that encodes a GA 2-oxidase, including, but not limited to: (1) sequences having at least 85% (preferably at least 90, 95, or as much as 100%) nucleotide sequence similarity with SEQ ID NO:57, 58, 60, 62, 64, 66, 67, 68, 69, 70, or 71; (2) sequences that encode a GA 2-oxidase having at least 70% (preferably at least 75, 80, 85, 90, 95, or as much as 100%) amino acid identity with an Arabidopsis GA 2-oxidase 4, an Arabidopsis 2-oxidase 5, a soybean GA 2-oxidase 1, a soybean GA 2-oxidase 2, a cotton GA 2 oxidase-1, a cotton GA 2 oxidase-2, a cotton GA 2 oxidase-3, a maize GA 2-oxidase 1, or a maize 2-oxidase 2.
According to another approach, such methods involve the use of transgenic plants having altered hormone levels resulting from a transgene that comprises a sequence that encodes an enzyme that metabolizes a precursor of the hormone to produce a metabolite that is not a precursor of the hormone in the transgenic plant. In the case of GA, such enzymes include phytoene synthases, C-20 oxidases, and 2xcex2,3xcex2-hydroxylases.
According to another aspect of the invention, related methods are provided that involve the use of transgenic plants (or seeds or seedlings thereof) that have a transgene that comprises a promoter (preferably a promoter that is preferentially expressed in developing seeds, during seed germination, or in early seedlings) and, operably linked to the promoter, a sequence that, when expressed, alters the level of an enzyme in the gibberellin biosynthetic pathway and causes an abnormal phenotype in the transgenic plant or the seed or seedling thereof (compared with a control). A phenotypically normal plant can be grown after applying to the plant or to a seed or seedling thereof a composition that comprises at least one GA compound, as defined herein. For example, GA levels can be affected by altering levels of a copalyl diphosphate synthase, a 3xcex2-hydroxylase, or a C-20 oxidase using a sequence that comprises: (1) at least 15 contiguous nucleotides of a member of the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 8; (2) a sequence having at least 85% (preferably at least 90, 95, or as much as 100%) nucleotide sequence identity with of a member of the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 8; or (3) a sequence that encodes a polypeptide having at least 70% (preferably at least 75, 80, 85, 90, 95, or as much as 100%) amino acid sequence identity with a polypeptide encoded by member of the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 8. Preferred GA compounds for rescuing normal plants in this case include ent-kaurene, ent-kaurenoic acid, ent-7xcex1-hydroxykaurenoic acid, steviol, GA12-aldehyde, GA12, GA15, GA24, GA9, GA53, GA44, GA19, GA20, GA5, GA4, GA7, GA3, and GA3-3-acetate, most preferably GA9, GA15, GA19, GA24, GA44, GA53, GA5, and steviol.
According to another aspect of the invention, additional related methods are provided that involve the use of transgenic plants (or seeds or seedlings thereof) that have a transgene that comprises a promoter (preferably a promoter that is preferentially expressed in developing seeds, during seed germination, or in early seedlings) and, operably linked to the promoter, a sequence that encodes an enzyme such as GA 2-oxidase that inactivates an endogenous GA, that is a GA that is normally present in the plant, causing at least one abnormal phenotype in the transgenic plant or the seed or seedling thereof (compared with a control). A phenotypically normal plant can be grown after applying to the plant or to a seed or seedling thereof a composition that comprises at least one GA compound that is metabolized by the seed or seedling to produce a product having gibberellin activity that is not degraded by the enzyme. In order to produce a GA 2-oxidase, such sequences include, for example: (1) sequences having at least 85% (preferably at least 90, 95, or as much as 100%) nucleotide sequence similarity with a member of the group consisting of SEQ ID NO:57, 58, 60, 62, 64, 66, 67, 68, 69, 70, and 71; and (2) sequences that encode a GA 2-oxidase having at least 70% (preferably at least 75, 80, 85, 90, 95, or as much as 100%) amino acid identity with an Arabidopsis GA 2-oxidase 4, an Arabidopsis 2-oxidase 5, a soybean GA 2-oxidase 1, a soybean GA 2-oxidase 2, a cotton GA 2 oxidase-1, a cotton GA 2 oxidase-2, a cotton GA 2 oxidase-3, a maize GA 2-oxidase 1, and a maize 2-oxidase 2. Preferred GA compounds include GA4, GA7, GA3, and GA3-3-acetate, most preferably GA3, and GA3-3-acetate.
According to another aspect of the invention, additional related methods are provided that involve the use of transgenic plants (or seeds or seedlings thereof) that have a transgene that comprises a promoter (preferably a promoter that is preferentially expressed in developing seeds, during seed germination, or in early seedlings) and, operably linked to the promoter, a sequence that encodes an enzyme that metabolizes a gibberellin precursor to produce a metabolite that is not a gibberellin precursor, for example a phytoene synthase, a C-20 oxidase, or a 2xcex2,3xcex2-hydroxylase, thereby reducing the level of a gibberellin and causing at least one abnormal phenotype in the transgenic plant or the seed or seedling thereof (compared to a control). A phenotypically normal plant can be grown after applying to the plant or to a seed or seedling thereof a composition that comprises at least one GA compound that substantially eliminates the abnormal phenotype. In the case of phytoene synthase, the preferred GA compounds are ent-kaurene, ent-kaurenoic acid, ent-7xcex1-hydroxykaurenoic acid, steviol, GA12-aldehyde, GA12, GA15, GA24, GA9, GA53, GA44, GA19, GA20, GA5, GA4, GA7, GA3, and GA3-3-acetate, most preferably GA9, GA15, GA19, GA24, GA44, GA53, GA5 and steviol. In the case of C-20 oxidase, the preferred GA compounds are GA9, GA4, GA20, GA1, GA7, GA3, and GA3-3-acetate, most preferably GA3 and GA3-3-acetate. In the case of 2xcex2,3xcex2-hydroxylase, the preferred GA compounds are GA9, GA41, GA53, GA44, GA19, GA20, GA15, GA7, GA3, and GA3-3-acetate, most preferably GA3 and GA3-3-acetate.
Compositions are provided that are useful in practicing the methods discussed above.
For example, nucleic acid segments are provided that comprise at least 12 contiguous nucleotides (and preferably at least 15, 18, 20, 24, 30, 40, or longer, up to and including the entire length) of a sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 8, 57, 58, 60, 62, 64, 66, 67, 68, 69, 70, 71, 75, 77, 79, and complements thereof, wherein the nucleic acid segment hybridizes specifically to the selected sequence under stringent hybridization conditions. Included are nucleic acid segments comprising sequences from SEQ ID NO:1, 2, 3, 4, 5, 6, or 8, that, when expressed in a plant cell (e.g., in an antisense orientation with respect to an operably linked promoter), reduce a level of an endogenous gibberellin compared with an otherwise identical plant cell in which the nucleic acid segment is not expressed.
In addition, nucleic acid segments are provided that comprise a sequence of at least 100 basepairs (and preferably at least 200, 300, 500, 700, 1000, or more) having at least 85% (and preferably at least 90, 95, or 100%) nucleotide sequence similarity with a member of the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 8, 57, 58, 60, 62, 64, 66, 67, 68, 69, 70, 71, 75, 77, 79, and complements thereof. Included among these nucleic acid segments are nucleic acid segments that encode a polypeptide with copalyl diphosphate synthase activity (SEQ ID NO:1, 2, 3, 4, and complements thereof), 3xcex2-hydroxylase activity (SEQ ID NO:5, 6, and complements thereof), C-20 oxidase activity (SEQ ID NO:8, 77, and complements thereof), GA 2-oxidase activity (SEQ ID NO: 57, 58, 60, 62, 64, 66, 67, 68, 69, 70, 71, and complements thereof), phytoene synthase activity (SEQ ID NO: 75 and complements thereof), and 2xcex2,3xcex2-hydroxylase activity (SEQ ID NO: 79 and complements thereof).
According to another aspect of the invention, nucleic acid constructs are provided that comprise a promoter that causes expression of an operably linked nucleic acid segment in a plant cell and, operably linked to the promoter, the nucleic acid segment comprising a sequence that encodes a polypeptide having a GA 2-oxidase activity. Expression of the nucleic acid segment in the plant cell results in inactivation of an endogenous gibberellin in the plant cell, thereby reducing levels of the endogenous gibberellin in the plant cell compared with an otherwise identical plant cell in which the nucleic acid segment is not expressed. For example, the sequence may encode a polypeptide having at least 70% amino acid sequence identity, preferably having only silently or conservative amino acid substitutions, and most preferably having 100% amino acid sequence identity with a polypeptide encoded by a member of the group consisting of SEQ ID NO:57, 58, 60, 62, 64, 66, 67, 68, 69, 70, 71, and complements thereof.
According to another aspect of the invention, nucleic acid constructs are provided that comprise a promoter that causes expression of an operably linked nucleic acid segment in a plant cell and, operably linked to the promoter, the nucleic acid segment comprising a sequence that encodes a polypeptide having a phytoene synthase, C-20 oxidase, or 2xcex2,3xcex2-hydroxylase activity. Expression of the nucleic acid segment in the plant cell results in metabolism of a gibberellin precursor in the plant cell to produce a metabolite that is not a gibberellin precursor in the plant cell, thereby reducing levels of the endogenous gibberellin in the plant cell compared with an otherwise identical plant cell in which the nucleic acid segment is not expressed. For example, the sequence may encode a polypeptide having at least 70% amino acid sequence identity, preferably having only silently or conservative amino acid substitutions, and most preferably having 100% amino acid sequence identity with a polypeptide encoded by a member of the group consisting of SEQ ID NO: 75, 77, 79, and complements thereof.
According to another aspect of the invention, a promoter that is operable in plant cells is provided that comprises at least 15, preferably 25, 50, 100, 200, 300, 500, or 1000 contiguous nucleotides or more of SEQ ID NO:7. Such a promoter is preferably preferentially expressed in seedlings.
According to another aspect of the invention, transgenic plants are provided that comprise the nucleic acid segments, constructs, and promoters mentioned above. Preferably such transgenic plants are characterized by at least one phenotype selected from the group consisting of a shortened hypocotyl, shortened epicotyl, and both a shortened hypocotyl and shortened epicotyl compared with an otherwise identical plant that lacks the nucleic acid segment.
According to another aspect of the invention, compositions are provided that comprise a seed of a plant that has a gibberellin-deficiency that results in at least one abnormal phenotype in the seed or in a seedling of the plant compared with a seed or seedling of an otherwise identical plant having wild-type levels of gibberellin; and a composition applied to a surface of the seed that comprises an amount of at least one GA compound that is effective to substantially eliminate the abnormal phenotype. The seed may be of a non-transgenic plant (e.g., a GA-deficient point or deletion mutant) or a transgenic plant comprising a transgene comprising a promoter and, operably linked to the promoter, a sequence that, when expressed, reduces gibberellin levels in the seed or seedling. The GA compound is preferably selected from the group consisting of ent-kaurene, ent-kaurenoic acid, ent-7xcex1-hydroxykaurenoic acid, steviol, GA12-aldehyde, GA12, GA15, GA24, GA9, GA53, GA44, GA19, GA20, and GA5, most preferably GA9, GA15, GA19, GA24, GA44, GA53, GA5 and steviol.
According to another aspect of the invention, methods are provided for reversibly controlling morphology in a seedling of a transgenic plant in which the capacity to biosynthesize at least one plant hormone that affects normal morphology in said seedling is inhibited, resulting in a deficiency in the level of said plant hormone and modification of at least one morphological trait of the seedling compared to a control seedling. A substantially normal morphology is restored by contacting seed or seedling with an amount of at least one GA compound effective to restore substantially normal morphology, permitting the plants to be grown to maturity. For example, such methods are useful for controlling elongation of a seedling tissue or tissues such as hypocotyl, epicotyl, coleoptile, and/or plumule tissue in a seedling of the transgenic plant. In GA-deficient plants, for example, normal morphology can be restored to plants that display one or more of the following morphological traits: reduced emergence, inhibited shoot growth, reduced height or stature, reduced stem growth, etc.
According to another aspect of the invention, lodging is reduced or prevented in a plant that is susceptible to lodging under conditions that are conducive to lodging by methods that employing a transgenic plant wherein the capacity to biosynthesize one or more plant hormones that affects the height of the seedling or plant is inhibited, resulting in a deficiency in the level of the hormone(s) and reduced height, compared to a control. After the conducive conditions are no longer present, the plant or a seed or seedling thereof can be grown to a normal height by contacting seed of the plant with an amount of at least one GA compound that is effective to increase the height of the seedling or plant.